When a wound occurs, several changes take place to minimize blood loss, referred to as the hemostatic response. First, the blood vessels slow the flow of blood past the wound site. Next, platelets that circulate in the blood are collected at the wound site to form a plug. Finally, a second system (the coagulation cascade), based upon the action of multiple proteins (called clotting factors) that act in concert to produce a fibrin clot is activated. These two systems (also known by the terms “primary hemostasis” and “secondary hemostasis”) work in concert to form a clot. Disorders in either system can yield conditions that cause either too much or too little clotting.
Platelets serve three primary functions. When a break in a blood vessel occurs, substances are exposed that normally are not in direct contact with the blood flow. These substances (primarily collagen and immobilized von Willebrand factor) allow the platelets to adhere to the broken surface (1st function). Once a platelet adheres to the surface, it releases chemicals that attract additional platelets to the damaged area, referred to as platelet aggregation (2nd function). These two processes are the first responses to stop bleeding. The platelet aggregates then serve as support for the processes of the coagulation cascade (3rd function). The protein based system (the coagulation cascade) serves to stabilize the clot that has formed and further seal up the wound.
The goal of the coagulation cascade (the “secondary hemostasis”) is to form a fibrin mesh within the platelet aggregate to stabilize the clot. All of the clotting factors have an inactive and an active form. Once activated, the factor will serve to activate the next factor in the sequence until fibrin is formed.
The many reactions in the coagulation cascade that are necessary to form a clot are described [Hematology—basic principles and practice, editors: Hoffman R, Benz E J, Shattil S J, Furie B, Cohen H J, Silberstein L E Churchill Livingstone 1995, P-1569]
The coagulation cascade is a chain of enzymatic reactions leading to the formation of a fibrin (F) clot [Platelets, Editor Michelson A D, Academic Press 2002, P-230]. The physiological cascade is initiated at the extrinsic pathway, by the exposure of tissue factor (TF), at the site of vessel injury or on activated blood cells (endothelial cells, monocytes etc). Tissue factor forms a complex with factor FVII, converting to it's activated form (FVIIa). This complex is then capable of activating factors X (FX), by creating a complex known as the “extrinsic tenase”), and factor IX (FIX). These activated factors (FIXa and FXa), then continue the enzymatic cascade at both the intrinsic (FIXa) and the common (FXa) pathways. Factor Xa forms a complex with prothrombin (PT), activated factor V (FVa) and phospholipids (derived from activated platelets), in the presence of calcium (this complex is termed “prothrombinase”), leading to activation of prothrombin to thrombin (T). Thrombin is a key player in the coagulation cascade, promoting many steps including: a) cleaving of fibrinogen (FG) to fibrin (F) monomers, which then spontaneously form polymers, b) activating factor XIII which cross-links fibrin polymers to create an insoluble fibrin clot, c) promoting the intrinsic cascade in a positive feedback loop by activating FXI, FVIII and FV, and d) activating platelets.
The intrinsic pathway is initiated by the activation of Factor XI (FXI) leading to activation of FIX, which then forms a complex with FX, Factor VIIIa (FVIIIa) and phospholipids, in the presence of calcium (known as the “intrinsic tenase” complex). This complex leads to the activation of Factor X (FXa), which then continues the reaction to the prothrombinase step (the common pathway).
The coagulation cascade is controlled by natural anticoagulants that inhibit different steps of the reaction. Thus anti-thrombin III (ATIII), inhibits mostly thrombin and Factor Xa, by creating a complex that prevents their enzymatic function. This effect is mediated by heparin, which activates ATIII and assembles the two proteins, allowing a better anticoagulant effect.
Another anticoagulant pathway includes protein C (PC) and protein S (PS), cooperatively capable of cleaving Factor V and Factor VIII, thus inhibiting the tenase and the prothrombinase steps.
Activated platelets express on their surface negatively charge phospholipids, that allow the assembly of coagulation factors leading to the creation of these complexes. The formation of these complexes (the intrinsic and extrinsic tenase as well as the ptothrombinase), increase significantly the rate of the coagulation reaction, by enhancing the interaction of the closely assembled clotting factors on the platelet membrane. Thus physiological clot formation is produced mostly on the surface of activated platelets, which are the first to adhere and aggregate at the site of the injured vessel wall.
The different factors involved in clot formation may be categorized as those which facilitate clot formation and those which block clot formation.
Blood clotting disorders may be divided into those related to platelet disorders, and those related to coagulation cascade disorders. If the clotting system can not adequately form clots, then the result is a bleeding disorder (hemophilia); if the clotting system forms clots too easily, then the result is formation of excess clots (thrombophilia).
Platelet disorders occur when there are too few platelets, too many platelets or a normal number of platelets that do not function in the normal manner. Having too few platelet or platelets that do not function well (for example, aspirin use) can lead to a bleeding tendency. Likewise, too many platelets can predispose to a tendency to clot excessively.
The coagulation cascade also has the potential to cause both an inability to form clots (hemophilia) and an excessive ability to form clots (thrombophilia). Hemophilic states result when there are decreased levels of the clotting factors. There are two primary disorders, hemophilia A and hemophilia B. Hemophilia A results from low levels of factor VIII and hemophilia B results from low levels of factor IX. Low levels of virtually any of the factors (with the exception of factor XII) will result in an inability to form blood clots; and thus, excess bleeding.
Hereditary bleeding disorders other than hemophilia may occur due to deficiencies or defects in other specific clotting factors. They are diagnosed by specific laboratory studies and treated by factor replacement.
Deficiencies in other factors, such as V, VII, X, and XIII, are rarer than the hemophilias, but produce similar symptoms. Factor XI deficiency is a common bleeding disorder among Ashkenazic Jews' affecting about 10% of them. Diagnosis depends on detection of bleeding symptoms, family history, and laboratory testing. Treatment usually involves blood transfusions, especially replacement of the missing coagulation factor, medications such as desmopressin (DDAVP), and antifibrolytic medications such as ε-aminocaproic acid (Amicar), and tranexamic acid (TA).
Hereditary von Willebrand's disease is an inherited bleeding disorder that affects the von Willebrand factor (vWf), which is necessary for platelet function and for fibrin clot formation. In von Willebrand's disease, there is a defect in the body's ability to produce vWf. This results in excessive bleeding from the mucus membrane of body cavities, such as the nose, the uterus, the bladder, and the rectum.
Another coagulation disorder is dysfibrinogenemia. Fibrinogen is the molecule that eventually becomes a fibrin, the major blood clotting protein. Fibrinogen is produced in the liver and bone marrow and released into the blood and eventually becomes fibrin. Dysfibrinogenemia is produced by an inherited problem in fibrinogen production. Abnormal fibrinogen is produced, leading to clotting problems in some individuals.
U.S. Pat. No. 5,523,238 describes a method and apparatus for determining platelet function in primary hemostasis. The method comprises obtaining a whole blood sample or platelet containing fraction of the blood (optionally mixed with an anticoagulant) and introducing the sample into a vessel having a flat bottom, the inner surface of which is covered with a substrate capable of inducing platelet adhesion to the surface and aggregation. The mixture inside the vessel is rotated such that a shear force is developed at the surface of the vessel. Then, parameters associated with primary hemostasis are determined. Such parameters include, inter alia, the amount of adhered platelets, aggregates size, aggregates' morphology, total area covered by the aggregates and size distribution of adhered platelets or aggregates.